ABCMdb

ABCC7 p.Lys978Cys

ClinVar:	c.2932A>T , p.Lys978* D , Likely pathogenic
Predicted by SNAP2:	A: D (71%), C: D (75%), D: D (91%), E: D (85%), F: D (85%), G: D (80%), H: D (71%), I: D (75%), L: D (80%), M: D (63%), N: D (71%), P: D (91%), Q: D (75%), R: D (66%), S: D (66%), T: D (75%), V: D (80%), W: D (91%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: D, V: D, W: D, Y: D,

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[hide] ATP-independent CFTR channel gating and allosteric... Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3. Wang W, Wu J, Bernard K, Li G, Wang G, Bevensee MO, Kirk KL
ATP-independent CFTR channel gating and allosteric modulation by phosphorylation.
Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3., 2010-02-23 [PMID:20133716]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.

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47 Cysteine modification experiments indicated that K978C is accessible to thiol-reactive compounds [e.g., the positively charged methanethiosulfonate reagent (MTSET)], which reversibly in- hibitedtheATP-independentcurrentsmediatedbythismutant(Fig. S2). Login to comment
48 For micropatches that contained sufficiently few K978C channels to resolve unitary currents, we observed that (i) the unitary currents (i.e., single-channel conductances) exhibited by the K978C construct were similar to those for WT-CFTR (Figs. S2 and S3); (ii) theK978Cconstructgateddynamicallyintheabsenceorpresenceof bath ATP (opened and closed spontaneously, albeit with somewhat longer openings than are typically observed for WT-CFTR (Figs. S2 and S3); and (iii) the primary effect of MTSET was to inhibit single-channel open probability (Po) by inhibiting the channel opening rate (Fig. S2). Login to comment
49 Table 1 compares the Pos, equilibrium gating constants, and Gibbs free energy differences for channel opening (ΔGos) for WT-CFTR channels and K978C-CFTR channels measured in the presenceand absenceofATP(examplerecords provided inFig.S3). Login to comment
51 The K978C mutant also had a higher Po in the presence of saturating ATP as compared with WT-CFTR. Login to comment
58 (B) K978C-CFTR macroscopic current across inside-out membrane patch excised from HEK-293T cell [ramp protocol (±80 mV); Methods]. Login to comment
71 (F) Very large ATP-independent current for K190C/K978C-CFTR. Login to comment
75 Impact of K978C mutation on open probability (Po), equilibrium gating constant (Keq), and Gibbs free energy difference for channel opening (ΔG) in the presence and absence of 1.5 mM ATP +ATP -ATP Po Keq ΔG, kJ/mol Po Keq ΔG, kJ/mol WT 0.38 ± 0.01 0.61 ± 0.02 1.2 ± 0.1 2.7 × 10-4 2.7 × 10-4 21 ± 0.8 ±8 × 10-5 ±8 × 10-5 K978C 0.84 ± 0.03* 6.53 ± 1.57* -4.3 ± 0.6 0.09 ± 0.01* 0.10 ± 0.02* 5.8 ± 0.5* Keq = Po/(1 - Po) and ΔG = -RT ln Keq where R is gas constant and T is temperature. Login to comment
77 Numbers of patches analyzed: three (WT, +ATP), six (WT, -ATP), six (K978C, +ATP), and five (K978C, -ATP). Login to comment
96 This initial observation was followed up by performing ATP titrations for the K978C-CFTR constitutive mutant and for WT-CFTR, which showed a nearly 10-fold decrease in the EC50 for ATP activation of the constitutive mutant (Fig. 2B and Fig. S1C). Login to comment
97 This large increase in ATP sensitivity of the K978C mutant is consistent with the predicted reciprocity between channel opening and ligand occupancy. Login to comment
100 The latter mechanism would be consistent with the elevated Po of the K978C mutant at saturating ATP (Fig. S3 and Table 1) and the slower deactivation of this construct following ATP removal. Login to comment
104 We were clued that ADP inhibits the ATP-independent activity of the constitutive mutants (e.g., K190C/K978C-CFTR channels) by the finding that their currents were lowest when hexokinase/glucose was added to induce current deactivation in the presence of 1.5 mM ATP (instead of perfusing ATP from the bath before adding the enzyme). Login to comment
106 Fig. S1D shows that the currents mediated by the K190C/K978C constitutive mutant increased on subsequent bath perfusion to remove the enzyme and all nucleotides and were reversibly inhibited by about 50% by adding back ADP alone. Login to comment
108 The constitutive activity of the K190C/K978C mutant was not inhibited by ADP when these loop mutations were introduced into a deletion construct that lacks NBD2 (Fig. S7 and Fig. 3). Login to comment
113 Fig. 3 shows that the K978C and K978S mutations markedly increased the macroscopic currents mediated by G551D-CFTR (the most common CF regulation mutant) and by Δ1198-CFTR (a deletion construct that lacks NBD2 and the carboxy terminal tail). Login to comment
117 Introducing the K978C or K978S mutation strongly enhanced the basal activities of G551D-CFTR and Δ1198-CFTR Fig. 2. Login to comment
119 (A) Slower deactivation of the K978C constitutive mutant following ATP removal by hexokinase/ glucose addition. Login to comment
121 (B) ATP titration curves for K978C-CFTR and WT-CFTR. Login to comment
123 Each symbol is the mean ± SEM for six (K978C) and eight (WT) experiments. Login to comment
124 Data were fit to the Michaelis-Menten equation; Km = 8.1 ± 1.4 μM and 65.2 ± 10.4 μM for K978C and WT, respectively. Login to comment
125 (C) Titration of ADP inhibition of K190C/K978C-CFTR current in the absence of ATP. Login to comment
128 The large basal currents mediated by K978C,S/ G551D and K978C,S/Δ1198 were ATP-independent but inhibited by CFTRinh-172 or glibenclamide. Login to comment
138 (C-E) High control currents for G551D and Δ1198-CFTR channels containing K978C or K978S mutations. Login to comment
146 The currents mediated by K978S/G551D and K978C/G551D were statistically greater than the G551D currents (P < 0.05, unpaired t test). Login to comment
153 In our whole-cell patch-clamp analysis of the K978C,S/G551D constructs, we observed that these mutants were strongly stimulated by a cAMP-activating mixture (Fig. 4A). Login to comment
155 To confirm and extend this point, we tested the PKA dependence of the activity of one of the constitutive mutants that lacks NBD2 (K978C/Δ1198) in excised membrane patches. Login to comment
160 We also introduced the double constitutive loop mutant (K190C/K978C) into a construct lacking both the R domain and NBD2 (Fig. 4E). Login to comment
164 First, the K978C loop mutation strongly increased the rate of activation at low doses of PKA (Fig. 4 F-H). Login to comment
167 This reciprocity is conceptually analogous to the observed reci- procitybetween constitutive activation bythe K978C mutation and the corresponding increase in ATP sensitivity (Fig. 2B). Login to comment
181 (A) Repre- sentativewhole-cellcurrent record showing strong stimulation of K978C/ G551D-CFTR by a forskolin-containing cAMP-activating mixture. Login to comment
183 (B) Representative current record showing PKA (110 U/ mL) activation of K978C/ Δ1198-CFTR in excised patch (1.5 mM ATP present initially). Login to comment
185 (C) No PKA stimulation of the current mediated by a corresponding construct lacking a large portion of the R domain, K978C/ΔR/Δ1198 [lacking residues 700-835 of the R domain (29)]. Login to comment
186 (D) Mean data showing relative activation of K978C/Δ1198-CFTR currents by PKA with or without the R domain (±SEMs, n = 7 and 13). Login to comment
187 (E) K190C/K978C/ΔR/Δ1198 channels are maximallyactiveunderbaselineconditions. Login to comment
189 (F and G) Representative records of PKA activation of K978C-CFTR and WT-CFTR, respectively. Login to comment
190 (H) Mean time course data for activation by 3 U/mL PKA (K978C) or 3-9 U/mL (WT). Login to comment
194 This conclusion is best supported by our finding that the activity of an NBD2-deletion construct that cannot be stimulated by ATP alone (K978C/Δ1198) is nonetheless strongly dependent on R domain phosphorylation. Login to comment
211 For the MTS (methanethiosulfonate) experiments (Fig. S2), the cysteine substitutions (e.g., K978C) were introduced into the C832A background because modification of C832 can affect CFTR currents (34). Login to comment

[hide] State-dependent regulation of cystic fibrosis tran... J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15. Wang G
State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3.
J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15., 2010-12-24 [PMID:20952391]

Abstract [show]
The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 x 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

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7 Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe3؉ no matter whether NBD2 was present or not. Login to comment
25 It has been demonstrated that K978C/P/S in CL3 promotes channel activity without ATP (17). Login to comment
97 Once the channel was open, Fe3ϩ could not exert any effect on hCFTR activity. Supporting this notion, a K978C mutant from CL3 was also insensitive to Fe3ϩ for a limited time because of a high open probability of 0.84 (17). Login to comment
98 As shown in Fig. 2B, the K978C mutant channel exhibited spontaneous activity when excised from the HEK-293T cell but the resulting current ran down by an unknown mechanism. Login to comment
100 A similar result was also found in a K978C-⌬1198 construct (Fig. 2C). Login to comment
114 Once K978C reduced the activation energy for channel opening, Fe3ϩ had no influence on channel activity. Login to comment
149 Macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing the hCFTR construct (A) and the K978C mutant (B). Login to comment
151 The arrows indicate the final concentrations. C, fractional inhibition of the current by Fe3ϩ for mutants K978C and K978C/ ⌬1198 (n ϭ 3-9). Login to comment
215 Once a K978C- or AMP-PNP-induced decrease in the activation energy overcomes an energy barrier caused by the interfacial Fe3ϩ bridge, channel activity will no longer be modulated by the metal bridge (Fig. 8B). Login to comment

[hide] The inhibition mechanism of non-phosphorylated Ser... J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8. Wang G
The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8., 2011-01-21 [PMID:21059651]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.

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22 Our recent study also demonstrated that a K190C/S mutation from CL1 enhances ATP-independent channel opening induced by a K978C/P/S mutation from CL3 (17). Login to comment
185 More importantly, H950R/S768R completely removed PKA dependence of channel activity (Fig. 7, A and D) no matter whether K978C, which promotes the channel opening without ATP, was inserted and accelerated channel activation by ATP (Fig. 7B) or not. Login to comment
255 PKA-dependent activity of His950 and Ser768 mutants with different H-bond donors and acceptors. A and B, activation of H950R/S768R (A) and H950R/S768R/K978C (B) before and after ATP (1. Login to comment

[hide] A unified view of cystic fibrosis transmembrane co... J Biol Chem. 2011 Apr 15;286(15):12813-9. Epub 2011 Feb 4. Kirk KL, Wang W
A unified view of cystic fibrosis transmembrane conductance regulator (CFTR) gating: combining the allosterism of a ligand-gated channel with the enzymatic activity of an ATP-binding cassette (ABC) transporter.
J Biol Chem. 2011 Apr 15;286(15):12813-9. Epub 2011 Feb 4., 2011-04-15 [PMID:21296873]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ion channel in that its gating is coupled to an intrinsic enzymatic activity (ATP hydrolysis). This enzymatic activity derives from the evolutionary origin of CFTR as an ATP-binding cassette transporter. CFTR gating is distinct from that of a typical ligand-gated channel because its ligand (ATP) is usually consumed during the gating cycle. However, recent findings indicate that CFTR gating exhibits allosteric properties that are common to conventional ligand-gated channels (e.g. unliganded openings and constitutive mutations). Here, we provide a unified view of CFTR gating that combines the allosterism of a ligand-gated channel with its unique enzymatic activity.

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134 The strongest evidence for the latter is the observation that CFTR channels that lack NBD2 but possess one of the constitutive loop mutations (e.g. CFTR-K978C/⌬1198) are strongly stimulated by PKA phosphorylation of the R domain even 4 Regarding Fig. 4A, it is important to note that we do not consider the open pore conformation at the TMDs (whatever that looks like structurally) to be strictly demanded by a tight NBD1-NBD2 dimer, nor do we consider the closed pore conformation to be demanded by the absence of the NBD1-NBD2 dimer. Login to comment

[hide] Thermally unstable gating of the most common cysti... J Biol Chem. 2011 Dec 9;286(49):41937-48. Epub 2011 Sep 30. Wang W, Okeyo GO, Tao B, Hong JS, Kirk KL
Thermally unstable gating of the most common cystic fibrosis mutant channel (DeltaF508): "rescue" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops.
J Biol Chem. 2011 Dec 9;286(49):41937-48. Epub 2011 Sep 30., [PMID:21965669]

Abstract [show]
Most cystic fibrosis (CF) cases are caused by the DeltaF508 mutation in the CF transmembrane conductance regulator (CFTR), which disrupts both the processing and gating of this chloride channel. The cell surface expression of DeltaF508-CFTR can be "rescued" by culturing cells at 26-28 degrees C and treating cells with small molecule correctors or intragenic suppressor mutations. Here, we determined whether these various rescue protocols induce a DeltaF508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on DeltaF508-CFTR function. Low temperature-rescued DeltaF508-CFTR channels irreversibly inactivated with a time constant of 5-6 min when excised patches were warmed from 22 degrees C to 36.5 degrees C. A panel of CFTR correctors and potentiators that increased DeltaF508-CFTR maturation or channel activity failed to prevent this inactivation. Conversely, three suppressor mutations in the first nucleotide binding domain rescued DeltaF508-CFTR maturation and stabilized channel activity at 36.5 degrees C. The constitutive loop mutations increased ATP-independent activity of low temperature-rescued DeltaF508-CFTR but did not enhance protein maturation. Importantly, the ATP-independent activities of these DeltaF508-CFTR constructs were stable at 36.5 degrees C, whereas their ATP-dependent activities were not. Single channel recordings of this thermally stable ATP-independent activity revealed dynamic gating and unitary currents of normal amplitudes. We conclude that: (i) DeltaF508-CFTR gating is highly unstable at physiologic temperature; (ii) most rescue protocols do not prevent this thermal instability; and (iii) ATP-independent gating and the pore are spared from DeltaF508-induced thermal instability, a finding that may inform alternative treatment strategies.

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7 Here, we determined whether these various rescue protocols induce a ⌬F508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ⌬F508-CFTR function. Login to comment
52 In a recent study we showed that two cytosolic loop mutations (e.g. K190C and K978C) promote ATP-independent CFTR channel activity, allosterically increase ATP and PKA sensitivity, and also significantly restore the function of mutant CFTR channels that cannot be activated by ATP binding or NBD dimerization (e.g. G551D and ⌬1198, which lacks NBD2) (47). Login to comment
57 The cytosolic loop mutations (K978C and K190C/K978C) do not improve ⌬F508-CFTR processing but do increase the ATP-independent channel activity of ⌬F508 channels that are delivered to the cell surface by incubation at low temperature. Login to comment
64 K978C and K190C/K978C mutations, which promote ATP-independent channel activity (47), were introduced into the ⌬F508-CFTR construct. Login to comment
162 Constitutive Loop Mutations Increase ATP-independent Channel Activity of ⌬F508-CFTR, Which Is More Stable at Physiologic Temperature-Previously, we showed that several mutations in intracellular loop1 and loop3 (e.g. K190C and K978C) increase the ATP-independent channel activities of several CFTR constructs, including mutant forms that cannot be activated by ATP (e.g. G551D and ⌬1198-CFTR, an NBD2 deletion mutant) (47). Login to comment
164 Fig. 6A shows that neither K978C nor K190C/K978C improved the maturation of ⌬F508-CFTR when cells were grown either at 37 °C or 27 °C, as determined by immunoblotting. Login to comment
169 Fig. 7A shows that K978C/⌬F508-CFTR exhibited a partial decrease of current when the bath temperature was elevated to 36.5 °C, but on average 30-40% of this current remained after warming the bath (see Fig. 7D). Login to comment
170 Interestingly, for approximately 50% of the patches the current slightly recovered when the temperature returned to room temperature, indicating that some of the current decrease for K978C/⌬F508-CFTR was reversible. Login to comment
171 These results indicate that K978C partially protects ⌬F508 channel activity from thermal inactivation. Login to comment
172 We then added hexokinase/glucose to remove ATP to determine whether the remaining current is ATP-independent. As shown in Fig. 7A, most of the current that remained following warming the bath was insensitive to removal of ATP, indicating that the ATP-independent channel activity of K978C/⌬F508-CFTR is far more stable at 36.5 °C compared with that of ⌬F508-CFTR alone (Fig. 1B). Login to comment
173 To follow up this result we examined the thermal stability of the K190C/K978C/⌬F508 construct that has a greater relative ATP-independent channel activity. Login to comment
174 Fig. 7B shows that the K190C/K978C double mutation greatly protected the channel activity from thermal inactivation with nearly all of the remaining current following warming the bath being ATP-independent. As a control we also tested the thermal stability of the ATP-independent channel activity of G551D-CFTR channels with the K978C mutation. Login to comment
175 Fig. 7C shows that raising the temperature did not inactivate the K978C/G551D-CFTR current, indicating that the ATP-independent channel activity of K978C/G551D is thermally stable. Login to comment
185 Error bars, S.E. Thermal Instability of ⌬F508-CFTR Gating DECEMBER 9, 2011•VOLUME 286•NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 41943 the ATP-independent channel activity of K978C/⌬F508-CFTR and to a greater extent K190C/K978C/⌬F508-CFTR was resistant to temperature-induced inactivation. Login to comment
188 To examine this idea further we recorded the unitary currents mediated by single K978C/⌬F508-CFTR channels in micropatches obtained with small tip pipettes. Login to comment
190 Fig. 8E shows that the amplitudes of the singlechannelcurrentsforK978C/⌬F508afterincubationat36 °C were similar to those for WT- and K978C-CFTR (47), indicating that the conformation of the pore for K978C/⌬F508 is not obviously compromised at physiologic temperature. Login to comment
195 We found that constitutive mutations in cytosolic loop1 and loop3 (i.e. K978C and K190C/K978C) increased ATP-independent channel activity of low temperature-rescued ⌬F508-CFTR. Login to comment
199 FIGURE6.Cytosolicloopmutations(K978CandK190C/K978C)increaseATP-independentchannelactivityoflowtemperature-recued⌬F508-CFTR.A, immunoblot showing that loop mutations do not improve maturation of ⌬F508 at 27 °C. B-D, ATP-dependent channel activities of ⌬F508, K978C/⌬F508, and K190C/K978C/⌬F508 revealed by adding hexokinase/glucose to scavenge ATP. Login to comment
201 E, mean fractional ATP-independent currents for ⌬F508 (n ϭ 6), K978C/⌬F508 (n ϭ 5), and K190C/K978C/⌬F508-CFTR (n ϭ 5). Login to comment
218 K978C/⌬F508 and K190C/K978C/⌬F508-CFTR are more stable at physiologic temperature. Login to comment
219 A, effect of raising temperature on K978C/⌬F508. Login to comment
221 B, effect of raising temperature on K190C/K978C/⌬F508-CFTR, which is more thermally stable. Login to comment
222 C, effect of raising temperature on K978C/G551D-CFTR. Login to comment
223 D, mean fractional current decrease for ⌬F508 (data from Fig. 1D), K978C/⌬F508, K190C/K978C/⌬F508, and K978C/ G551D. Login to comment
237 A and B, effect of raising temperature on ATP-independent currents of K978C/⌬F508- and K190C/K978C/⌬F508-CFTR. Login to comment
239 C, mean fractional loss of ATP-independent current for K978C/⌬F508 and K190C/ K978C/⌬F508-CFTR (n ϭ 4 for each construct). Login to comment
240 D, effect of raising temperature on unitary currents of K978C/⌬F508. Login to comment
242 Effect of raising bath temperature on K978C/⌬F508 current was monitored at the macroscopic level (ramp protocol), and then the unitary currents were recorded in the gap-free mode after reduction to room temperature. Login to comment

[hide] Conserved allosteric hot spots in the transmembran... J Biol Chem. 2014 Jul 18;289(29):19942-57. doi: 10.1074/jbc.M114.562116. Epub 2014 May 29. Wei S, Roessler BC, Chauvet S, Guo J, Hartman JL 4th, Kirk KL
Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.
J Biol Chem. 2014 Jul 18;289(29):19942-57. doi: 10.1074/jbc.M114.562116. Epub 2014 May 29., [PMID:24876383]

Abstract [show]
ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

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109 Fig. 1A also shows the location of a previously characterized GOF mutation (K978C) that locates near the base of TM9 in the outer TM collar (16). Login to comment
172 Additive GOF Effects of the P355A Mutation and a Mutation in the Outer TM Collar-We previously reported another class of GOF mutation that locates to the base of TM9 (Lys-978) in the putative outer TM collar that surrounds the principal pore-lining TMs (see Fig. 1A for predicted location of Lys-978 in CFTR structural models) (16). Login to comment
173 We reasoned that, given the different locations of the two classes of GOF mutations (pore versus outer collar), a double mutant (P355A/K978C) may exhibit an additive GOF effect. Login to comment
175 GOF mutations in the pore-lining TM6 (P355A) and in the outer TM collar (K978C) have additive effects on CFTR channel activity. Login to comment
176 A, macroscopic current record showing the relatively large ATP-independent current and robust activation by 2 mM AMP-PNP for the P355A/K978C double mutant.Fortherampprotocol,conditionswerethesameasforFigs.2-4.BandC,meanfractionalATP-freecurrentandrelativeAMP-PNPactivationnormalized to the control current at 1.5 mM ATP for the indicated single and double mutants. Login to comment
191 G, immunoblot of P355A-èc;1198, P355A-G551D, and P355A/K978C double mutants transiently expressed in HEK-293T cells. Login to comment
197 The double mutant (P355A/K978C-CFTR) exhibited relatively high ATP-independent currents in excised macropatches (b03;35% of the ATP control current) and strong activation by AMP-PNP that approached that by ATP (b03;90% of the ATP control current). Login to comment
199 It is also evident from the single mutant data in Fig. 5, B and C, that the K978C mutation had a somewhat stronger GOF effect than the P355A mutation. Login to comment
207 Introducing the P355A mutation increased the apparent ATP affinity of the Y1219G mutant (leftward shift in Fig. 6B). Login to comment
208 The K978C mutation in the outer TM collar (TM9) also increased the apparent ATP affinity of the Y1219G mutant and to a greater degree than the Pro-355 substitution, as would be expected if the former is a stronger GOF mutation (see also Fig. 5). Login to comment
210 These results also indicate that both classes of GOF mutations (Pro-355 in TM6; K978C in the outer TM collar) can compensate for a partial defect in ATP binding by allosteric coupling between the TMs and the NBDs (i.e. by "allosteric rescue" of the ATP binding defect). Login to comment
220 Curves, best fits to Hill equation with K values of 1522, 683, and 180 òe;M and Hill coefficients of 1.69, 1.93, and 1.72 for Y1219G (n afd; 6 patches), P355A/Y1219G (n afd; 5), and K978C/Y1219G (n afd; 4), respectively. Login to comment
284 GOF effects of the P355A and K978C mutations were additive, implying that they influence channel gating by different mechanisms. Login to comment
308 Both the P355A and K978C GOF mutations increased the ATP sensitivity of the Y1219G mutant with the K978C substitution restoring the ATP sensitivity of channel gating to nearly wild type levels. Login to comment
108 Fig. 1A also shows the location of a previously characterized GOF mutation (K978C) that locates near the base of TM9 in the outer TM collar (16). Login to comment
174 GOF mutations in the pore-lining TM6 (P355A) and in the outer TM collar (K978C) have additive effects on CFTR channel activity. Login to comment
190 G, immunoblot of P355A-èc;1198, P355A-G551D, and P355A/K978C double mutants transiently expressed in HEK-293T cells. Login to comment
196 The double mutant (P355A/K978C-CFTR) exhibited relatively high ATP-independent currents in excised macropatches (b03;35% of the ATP control current) and strong activation by AMP-PNP that approached that by ATP (b03;90% of the ATP control current). Login to comment
198 It is also evident from the single mutant data in Fig. 5, B and C, that the K978C mutation had a somewhat stronger GOF effect than the P355A mutation. Login to comment
209 These results also indicate that both classes of GOF mutations (Pro-355 in TM6; K978C in the outer TM collar) can compensate for a partial defect in ATP binding by allosteric coupling between the TMs and the NBDs (i.e. by "allosteric rescue" of the ATP binding defect). Login to comment
219 Curves, best fits to Hill equation with K values of 1522, 683, and 180 òe;M and Hill coefficients of 1.69, 1.93, and 1.72 for Y1219G (n afd; 6 patches), P355A/Y1219G (n afd; 5), and K978C/Y1219G (n afd; 4), respectively. Login to comment
283 GOF effects of the P355A and K978C mutations were additive, implying that they influence channel gating by different mechanisms. Login to comment
307 Both the P355A and K978C GOF mutations increased the ATP sensitivity of the Y1219G mutant with the K978C substitution restoring the ATP sensitivity of channel gating to nearly wild type levels. Login to comment

[hide] Long-range coupling between the extracellular gate... FASEB J. 2015 Nov 25. pii: fj.15-278382. Wei S, Roessler BC, Icyuz M, Chauvet S, Tao B, Hartman JL 4th, Kirk KL
Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.
FASEB J. 2015 Nov 25. pii: fj.15-278382., [PMID:26606940]

Abstract [show]
The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that: 1) all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy and 2) highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.-Wei, S., Roessler, B. C., Icyuz, M., Chauvet, S., Tao, B., Hartman, J. L., IV, Kirk, K. L. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

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201 To test this idea, the extracellular F337S mutation was combined with the previously characterized K978C mutation, which locates tothe cytosolic side below TM9 (14, 15). Login to comment
202 The K978C substitution increases ATP-free channel activity and enhances the ATP and PKA sensitivities of CFTR activation similar to that shown here for the F337S mutation (14). Login to comment
204 Removing bath ATP in the standard excised macropatch protocol decreased the currents mediated by F337S/K978C-CFTR by less than 20% (Fig. 10A, B). Login to comment
207 More strikingly, F337S/K978C-CFTR appeared to be nearly maximally active in the absence of both PKA and ATP (Fig. 10C-F). Login to comment
208 Substantial F337S/K978C-CFTR-mediated currents could be detected for macropatches that were excised in the absence of both PKA and ATP in Figure 6. Login to comment
220 In support of this interpretation, the Po of F337S/K978C-CFTR estimatedfrom multichannel records in theabsence ofbothPKAandATP(examplerecordinFig.10D)ranged from 0.34 to 0.95 with a mean Po (6SEM) of 0.53 6 0.08 (n = 7 patches;estimated using the conventional Clampfit protocol; see Methods). Login to comment
276 The F337S/K978C double mutant is nearly fully active in the absence of both exogenous PKA and ATP. Login to comment
277 A) Representative macroscopic current record showing that ATP removal by scavenger addition followed by bath perfusion with an ATP-free solution only modestly decreases the activities of F337S/K978C-CFTR channels. Conditions were identical to Fig. 4. Login to comment
282 C) Representative macroscopic current record showing that F337S/K978C-CFTR channels are nearly maximally active in excised patches in the absence of both ATP and exogenous PKA. Login to comment
284 D) Unitary current recording at a single holding potential for an excised patch containing 3 F337S/K978C-CFTR channels. Login to comment
288 E) Gap-free record at a single holding potential for an excised patch containing 80-100 F337S/K978C-CFTR channels showing substantial activity in the absence of bath PKA and ATP. Login to comment
289 F) Stationary noise plot for F337S/K978C-CFTR channels in the absence of PKA and ATP. Login to comment
329 Engineering a superactive CFTR The F337S/K978C double mutant has the highest single-channel activity in the absence of exogenous PKA and ATP of any CFTR construct that we have characterized to date. Login to comment
332 We anticipated that the F337S and K978C mutations would have additive GOF effects on ATP-free CFTR activity because they locate to opposite sides of the pore where they presumably impact CFTR structure in different ways. Login to comment
200 To test this idea, the extracellular F337S mutation was combined with the previously characterized K978C mutation, which locates tothe cytosolic side below TM9 (14, 15). Login to comment
203 Removing bath ATP in the standard excised macropatch protocol decreased the currents mediated by F337S/K978C-CFTR by less than 20% (Fig. 10A, B). Login to comment
206 More strikingly, F337S/K978C-CFTR appeared to be nearly maximally active in the absence of both PKA and ATP (Fig. 10C-F). Login to comment
219 In support of this interpretation, the Po of F337S/K978C-CFTR estimatedfrom multichannel records in theabsence ofbothPKAandATP(examplerecordinFig.10D)ranged from 0.34 to 0.95 with a mean Po (6SEM) of 0.53 6 0.08 (n = 7 patches;estimated using the conventional Clampfit protocol; see Methods). Login to comment
275 The F337S/K978C double mutant is nearly fully active in the absence of both exogenous PKA and ATP. Login to comment
281 C) Representative macroscopic current record showing that F337S/K978C-CFTR channels are nearly maximally active in excised patches in the absence of both ATP and exogenous PKA. Login to comment
283 D) Unitary current recording at a single holding potential for an excised patch containing 3 F337S/K978C-CFTR channels. Login to comment
287 E) Gap-free record at a single holding potential for an excised patch containing 80-100 F337S/K978C-CFTR channels showing substantial activity in the absence of bath PKA and ATP. Login to comment
328 Engineering a superactive CFTR The F337S/K978C double mutant has the highest single-channel activity in the absence of exogenous PKA and ATP of any CFTR construct that we have characterized to date. Login to comment
331 We anticipated that the F337S and K978C mutations would have additive GOF effects on ATP-free CFTR activity because they locate to opposite sides of the pore where they presumably impact CFTR structure in different ways. Login to comment

[hide] Converting nonhydrolyzable nucleotides to strong c... J Biol Chem. 2013 Jun 14;288(24):17122-33. doi: 10.1074/jbc.M112.442582. Epub 2013 Apr 25. Okeyo G, Wang W, Wei S, Kirk KL
Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations.
J Biol Chem. 2013 Jun 14;288(24):17122-33. doi: 10.1074/jbc.M112.442582. Epub 2013 Apr 25., [PMID:23620589]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5'-O-(thiotriphosphate) (ATPgammaS), adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPgammaS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPgammaS removal (taudeac approximately 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPgammaS activated even the strongest GOF mutant as strongly as ATP. ATPgammaS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPgammaS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.

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No. Sentence Comment
81 RESULTS A GOF Mutation Increases CFTR Activation by Poorly Hydrolyzable Nucleotides-Fig. 1 shows the strong activation of a previously characterized GOF mutant (K978C-CFTR) by AMP-PNP, ATPॹS, and GTPॹS in excised inside-out macropatches. Login to comment
83 Previously we showed that the K978C substitution increased the single channel open probabilities (Po) of phosphorylated channels in the absence of nucleotide by at least 2 orders of magnitude (13). Login to comment
84 This ligand-independent activity of K978C-CFTR is detected in excised macropatch recordings as a small current that persists after removal of bath ATP by a scavenger (hexokinase/glucose) and subsequent bath perfusion with ATP-free solution (Fig. 1A). Login to comment
87 In contrast, these analogs strongly increased the currents mediated by K978C-CFTR, although not to the level of saturating ATP (Fig. 1, A, C, and F and supplemental Fig. S2). Login to comment
94 Fig. 2 shows activation of the K978C GOF mutant by a saturating dose of ATPॹS (1.5 mM) at the single channel level. Login to comment
99 ATPॹS-activated K978C-CFTR channels opened and closed dynamically (i.e. were not "locked open") with a broad distribution of open durations that ranged from b0d;100 ms to occasional openings that lasted 10-20 s (Fig. 2B). Login to comment
104 Accordingly, we compared the activation by AMP-PNP of several previously described GOF CFTR mutants with increasing degrees of unliganded activity (K978P, K978C, and K190C/K978C; see Fig. 3). Login to comment
105 All three GOF mutants were more strongly activated by AMP-PNP than WT-CFTR, with the lowest and greatest relative activation observed for the weakest (K978P; Fig. 3, A, C, and D) and strongest (K190C/K978C; Fig. 3, B, C, and D) GOF mutant. Login to comment
110 In contrast, the K978C-CFTR currents that were activated by ATPॹS or AMP-PNP deactivated very slowly upon the removal of these agonists (Fig. 4, A-D). Login to comment
112 Poorly hydrolyzable nucleotides strongly activate the K978C-CFTR GOF mutant. Login to comment
113 A and B, representative excised inside-out macropatch records compare activation of K978C-CFTR (A) and WT-CFTR (B) by the indicated concentrations of AMP-PNP. Login to comment
118 Hexokinase/glucose was re-added after activation of K978C-CFTR by AMP-PNP followed by the addition of the CFTR-specific inhibitor, CFTRinh-172 (10 òe;M). Login to comment
119 The lower macroscopic control current for K978C-CFTR (PKA plus ATP) in panel A is due to somewhat lower expression of this GOF mutant compared with WT-CFTR and not to lower channelactivity.Infact,thismutanthadasubstantiallygreatersinglechannelPo thanWT-CFTRundercontrolconditionsandafterATPremoval(Ref.13andFig. Login to comment
121 C and D, representative excised inside-out macropatch records compare activation of K978C-CFTR (C) and WT-CFTR (D) by ATPॹS. Login to comment
128 Wild type channels also deactivated fairly slowly after ATPॹS removal but considerably faster than K978C-CFTR (Fig. 4, C and D). Login to comment
130 In this regard, we also observed that ATP-activated K978C-CFTR channels deactivated much slower than WT-CFTR when ATP was removed either by adding the hexokinase/glucose scavenger to the bath (13) or by bath perfusion (Fig. 4F). Login to comment
131 The deactivation time courses for ATPॹS-activated K978C-CFTR channels (b0e;100s) were considerably longer than the mean open times observed in the single channel experiments in Fig. 2 (b0d;5s). Login to comment
132 This disparity argues that K978C channels open and close dynamically even when ATPॹS is tightly bound. Login to comment
133 To explore this point further, we tracked the gating of K978C and wild type channels after ATPॹS removal in patches containing sufficiently few channels to permit detection of unitary events. Login to comment
134 K978C-CFTR channels continued to open and close up to several min after ATPॹS removal (Fig. 4E). Login to comment
136 The slow deactivation observed especially for K978C-CFTR channels implies that ATPॹS and AMP-PNP bind tightly, presumably at the NBD dimer interface (see below). Login to comment
140 Previously we reported that truncated channels that lack NBD2 but possess a GOF mutation (K978C/èc;1198-CFTR) exhibit detectable FIGURE 2. Login to comment
141 ATPॹS markedly increases the single channel open probability of K978C-CFTR. Login to comment
142 A, shown is a single channel record for excised inside-out patch containing one K978C-CFTR channel under control conditions (1.5 mM MgATP), after ATP removal with hexokinase/glucose, and after the addition of 1.5 mM ATPॹS.Channelswereprephosphorylatedwith110units/mlPKAfollowedbyPKIaddition.Holdingpotentialwasafa;60mV.Openingsaredownward.Measured Po values for each condition in this record are indicated. Login to comment
145 C, mean data (afe;S.E.; n afd; 3 patches) show that ATPॹS greatly increased the Po and single channel opening rate of K978C-CFTR. Login to comment
151 It should be noted that these truncated channels are not maximally active under the conditions of this experiment; i.e. previously we observed that K978C/èc;1198-CFTR channels are stimulated substantially by a compound that activates CFTR currents by an unknown mechanism (curcumin; Refs. 13 and 24; see also Fig. 5, E and F). Login to comment
162 In an earlier study we found that GOF mutations at residue 978 (K978C or K978S) promoted a substantial ATP-independent activity for the G551D mutant that could be augmented further by curcumin (13). Login to comment
163 Here we observed that K978C/G551D-CFTR channels could not be activated by ATPॹs (Fig. 5E) and instead were slightly but reproducibly inhibited by this nucleotide. Login to comment
169 A and B, representative macropatch records show AMP-PNP activation of K978P-CFTR (A) and K190C/K978C-CFTR (B). Login to comment
171 Note the previously described voltage-dependent rectification of K190C/K978C-CFTR currents (13). Login to comment
174 WT and K978C data are from Fig. 1F. Login to comment
182 Introduction of one of the GOF mutations into this CF mutant (K978C/G1349D-CFTR) substantially rescued its activity as evidenced by much higher ATP-dependent control currents and much lower relative activation by the potentiators (Fig. 6, B and C). Login to comment
183 K978C/G1349D-CFTR channels also exhibited detectable currents in the absence of any bath nucleotide as expected (Fig. 6D). Login to comment
184 Unlike K978C/G551D-CFTR channels, K978C/G1349D-CFTR channels remained sensitive to ATPॹS as did the G1349D mutant without the GOF substitution (Fig. 6, D-F). Login to comment
188 A, macropatch record shows slow deactivation of K978C-CFTR current after AMP-PNP washout. Login to comment
191 B, shown is slow deactivation of K978C-CFTR current after ATPॹS washout. Login to comment
192 C, WT-CFTR currents also deactivate fairly slowly after ATPॹS washout but faster than K978C-CFTR currents. Login to comment
194 D, mean deactivation time constants (afe;S.E.) were estimated from single exponential fits of currents after ATPॹS washout for WT-CFTR and K978C-CFTR (n afd; 6 each). Login to comment
196 By comparison, the mean deactivation time course for ATP-activated K978C-CFTR channels after washout of 1.5 mM ATP using the samerampprotocolwas51.0afe;3.3s(nafd;7).ThedeactivationtimecourseforATP-activatedwildtypechannelsafterATPremovalwastoorapidtoresolveusing this ramp protocol (i.e. WT-CFTR channels deactivate faster than the 10-s ramp period after ATP removal). Login to comment
197 E, micropatch experiments at a constant holding potential (afa;60 mV) show that ATPॹS-activated K978C-CFTR channels (top) and WT-CFTR channels (bottom) continue to open and close many seconds after removingATPॹSbybathperfusion(initiatedatthearrow).TheWT-CFTRpatchwasobtainedusingalargertippipettetooptimizedetectionofATPॹS-activated channels. Login to comment
199 Time to full deactivation for K978C-CFTR channels after ATPॹS removal in these micropatch experiments ranged from 19 to 246 s. Login to comment
200 F, control micropatch experiments (also at afa;60 mV) show more rapid deactivation of WT-CFTR channels and K978C-CFTRchannelsafterremovalofATPbybathperfusion.Thesepatcheswereobtainedusingsmallertippipettestodetectunitarycurrentsinthepresence of 3 mM ATP. Login to comment
201 The slower deactivation of K978C-CFTR channels after ATP removal is consistent with the slower macroscopic deactivation time course reported previously (Ref. 13; see also "Discussion"). Login to comment
210 Fourth, poorly hydrolyzable nucleotides are weaker CFTR activators than ATP even when they apparently bind tightly to the NBDs, as was the case for the K978C GOF mutant. Login to comment
217 Perturbation of the NBD dimer interface inhibits K978C-CFTR activation by AMP-PNP and ATPॹS. Login to comment
219 B, no activation of the K978C/èc;1198 NBD2 deletion mutant by AMP-PNP added to final concentrations (in mM) of 0.1, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, and 3.0 is shown. Note that the control current mediated by this construct is entirely ATP-independent (e.g. no effect of hexokinase(Hex)/glucose).C,diamide(Dia)/glutathione(20òe;M each)slowlyinhibitstheK978P-CFTRcurrentactivatedby2mM AMP-PNP.Subsequentaddition of 5 mM DTT reversed this inhibition. Login to comment
222 E, no activation of K978C/G551D-CFTR by 1.5 mM ATPॹS is shown. Note subsequent strong activation by 30 òe;M curcumin. Login to comment
224 F, very small activation of K978C/G551D-CFTR by 2 mM AMP-PNP is shown. Login to comment
226 were activated by these analogs precluded performing quantitative titrations like we performed for the K978C GOF mutant. Login to comment
228 Our results also indicate that, at least for the K978C GOF mutant, these analogs are less effective than ATP at promoting channel opening at saturating concentrations when they apparently bind tightly at the NBD dimer interface. Login to comment
231 In agreement with this, ATPॹS-activated K978C-CFTR channels opened and closed on a much faster time scale (i.e. they were not locked open) than they deactivated upon ligand removal. Login to comment
233 K978C channels also were observed to open and close several minutes after removing the ATPॹS (Fig. 4), which supports the view that the opening and closing of ATPॹS-activated channels is not tightly coupled to ATPॹS binding and unbinding (although interpreting these nonsteady-state single channel experiments is complicated by the fact that we cannot distinguish di-liganded channels from mono-liganded channels that have unbound one ligand molecule; see also below). Login to comment
236 Rescue of G1349D-CFTR gating by K978C and differential activation of this NBD2 mutant by ATPॹS and AMP-PNP. Login to comment
238 B, a corresponding macropatch record for K978C/G1349D-CFTR shows large control current and relatively small activation by NPPB-AM and curcumin. Login to comment
239 C, shown are mean control currents (1.5 mM ATP) and mean -fold activation by the combination of curcumin and NPPB-AM for G1349D-CFTR and K978C/G1349D-CFTR (n afd; 6-17; **, p b0d; 0.01 compared with G1349D). Login to comment
240 D and E, representative macropatch records show much smaller activation of K978C/G1349D-CFTR or G1349D-CFTR current by 2 mM AMP-PNP versus 1.5 mM ATPॹS. Hex, hexokinase. Login to comment
259 In this regard, the substitutions at position Lys-978 that had the strongest GOF effects (K978C, K978S, and K978P; Ref. 13) also are predicted to have the greatest disruptive effects on the presumed helical structure of cytosolic loop3 and TM9 based on secondary structure predictions (results not shown). Login to comment
265 The slower deactivation (and presumably slower unbinding) after ATPॹS removal that we observed for the K978C GOF mutant relative to wild type CFTR (Fig. 4) is consistent with the latter mechanism. Login to comment
271 In addition, ATPॹS was effective at much lower concentrations, and unlike the case for AMP-PNP, the ATPॹS titration curve for K978C-CFTR activation could be fit only by a two-binding site model with different affinities. Login to comment
282 The notion that AMP-PNP might be functionally effective primarily at site 1 in the absence of ATP would be consistent with the stronger effect of the NBD2 signature sequence mutation (nearest site 1) on AMP-PNP activation of K978C-CFTR. Login to comment
289 Previously we showed that the activity of the severely defective G551D mutant was increased substantially by GOF mutations such as K978C (13). Login to comment
291 In contrast, we observed a more authentic rescue for the G1349D- K978C construct in the form of much greater ATP-dependent currents and lower relative activation by potentiators. Login to comment
292 This construct also exhibited larger ATP-independent currents, as expected for channels with the K978C GOF mutation. Login to comment
294 G1349D-CFTR rescue by the K978C GOF mutation is analogous to its enhancement of CFTR activation by ATPॹS or AMP-PNP. Login to comment

[hide] An electrostatic interaction at the tetrahelix bun... J Biol Chem. 2014 Oct 31;289(44):30364-78. doi: 10.1074/jbc.M114.595710. Epub 2014 Sep 4. Wang W, Roessler BC, Kirk KL
An electrostatic interaction at the tetrahelix bundle promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator (CFTR) channel opening.
J Biol Chem. 2014 Oct 31;289(44):30364-78. doi: 10.1074/jbc.M114.595710. Epub 2014 Sep 4., [PMID:25190805]

Abstract [show]
The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.

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No. Sentence Comment
11 ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Login to comment
12 Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge-reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. Login to comment
141 Combining E267 and K1060 Charge-reversal Mutations with a Gain of Function (GOF) Mutation Reveals the Importance of the Tetrahelix Bundle in Mediating Channel Opening in the Absence of ATP Binding or NBD Dimerization-Previously we reported a class of GOF mutation in ICL3 near the base of TM9 that increases both ATP-dependent and ATP-independent channel activity (e.g. K978C (12)). Login to comment
144 Regarding the first issue, Fig. 6A shows that introducing the K978C substitution into the E267R mutant substantially restored ATP-dependent channel activity (compare with Fig. 2B). Login to comment
145 The E267R/K978C double mutant exhibited normalized control currents and potentiator responses in macropatch experiments that were similar to wild type levels (see legend for mean data). Login to comment
146 This rescue of the ATP-dependent activity of the E267 charge-reversal mutant is perhaps not surprising given that the K978C substitution reduces the free energy difference FIGURE 2. Login to comment
171 Regarding the second issue, Fig. 6, B-E show that the E267R and K1060E mutations reduced the ATP-free activity of the K978C GOF mutant. Login to comment
174 The latter interpretation is further supported by the results in Fig. 7, which show that the E267R mutation also reduced the ATP-free channel activity of K978C/èc;1198-CFTR, a truncation mutant lacking NBD2 but possessing the indicated GOF mutation. Login to comment
262 Disrupting the E267-K1060 Interaction Primarily Impacts Channel Opening, not Burst Duration or NBD Dimer Stability-The charge-reversal mutations at these two positions dramatically decreased the macroscopic currents and apparent single FIGURE 6. Interplay between the charge-reversal mutations at the bundle interface and a previously reported GOF mutation (K978C); evidence that the E267RandK1060EmutationsalsoinhibitATP-freechannelactivity.A,E267R/K978CdoublemutantbehavesmorelikewildtypechannelsinthepresenceofATP inmacropatchexperiments.ConditionswereidenticaltoFig.2A.Themeanpercentcontrolcurrentsofthisdoublemutantwhennormalizedtothemaximalcurrents measuredafterpotentiatoradditionwere94afe;5.4%and82.8afe;3.9%beforeandafterPKIaddition,respectively(nafd;5).Thesevaluesarenotsmaller(infact,somewhat larger) than those for WT-CFTR (see Fig. Login to comment
264 B-D, macroscopic records for the indicated K978C single and double mutants showing that the E267R and K1060E substitutions decreased the fractional currents remaining after ATP removal. Login to comment
266 E267R/K978C-CFTR was activated strongly by the subsequent addition of 2 mM AMP-PNP in the absence of ATP, as reported previously for the K978C GOF mutant (panels B, C and Ref. 47). Login to comment
280 A, control macroscopic record showing the substantial control current and correspondingly modest stimulation by potentiator addition for the previously described K978C/èc;1198 truncation mutant lacking NBD2. Login to comment
282 As reported earlier (12), the channel activity of this construct is ATP-independent and is attributable to the presence of the K978C GOF mutation. Login to comment
289 **, p b0d; 0.01 compared with K978C/èc;1198 by unpaired t test. N is indicated in parentheses. CFTR Gating Mechanism 30374 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, tion between inactive and active conformations in response to ATP binding and NBD dimerization. Login to comment
290 The E267-K1060 Interaction Also Promotes CFTR Channel Opening in the Absence of ATP Binding or NBD Dimerization- Combining a previously reported GOF mutation (K978C) with the E267R or K1060E bundle mutations revealed that the latter mutations also inhibit ATP-free channel activity both in the presence and absence of NBD2 (i.e. for the èc;1198-CFTR truncation construct). Login to comment
291 The K978C substitution did restore nearly normal ATP-dependent activity of the E267R mutant as evidenced by high control currents in the presence of ATP and PKA and small relative activation by potentiators. Login to comment
293 Apparently the favorable effect of the K978C mutation on the energetics of channel opening is sufficient to overcome the negative impact of the bundle mutation on gating when ATP binding and NBD dimerization can occur. Login to comment
294 Conversely, each of the E267R and K1060E mutations substantially reduced the fractional ATP-independent currents exhibited by the full-length K978C-CFTR construct and also reduced the control currents and increased the relative stimulation by potentiators for the NBD2-deletion construct bearing the GOF mutation (K978C/ èc;1198-CFTR). Login to comment
298 According to this view, GOF mutations such as K978C enhance the channel activity of the èc;1198-CFTR construct lacking NBD2 by allosterically promoting the E267-K1060 interaction at the bundle interface. Login to comment
301 The latter mechanism is supported by our earlier observation that PKA phosphorylation of the R domain markedly increases the channel activity of the NBD2 deletion construct, K978C/èc;1198-CFTR (12). Login to comment
326 CFTR Gating Mechanism 30376 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 44ߦOCTOBER 31, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, observed that the K978C GOF mutation in ICL3 near the base of TM9 increased the PKA sensitivity of channel activation even though this residue is nowhere near a PKA phosphorylation site in the polypeptide sequence (12). Login to comment

[hide] Functional Architecture of the Cytoplasmic Entranc... J Biol Chem. 2015 Jun 19;290(25):15855-65. doi: 10.1074/jbc.M115.656181. Epub 2015 May 5. El Hiani Y, Linsdell P
Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.
J Biol Chem. 2015 Jun 19;290(25):15855-65. doi: 10.1074/jbc.M115.656181. Epub 2015 May 5., [PMID:25944907]

Abstract [show]
As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm.

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112 We did not observe any ATPand PKA-dependent, CFTRinh-172-sensitive macroscopic currents in inside-out patches associated with R153C, H949C, or K978C (Fig. 3), most likely due to lack of channel expression in the membrane (11). Login to comment